The multi-hit hypothesis in basal-like breast cancer

Abstract

It has been known for many years that for a "normal" un-transformed cell to become immortal and subsequently tumorigenic requires multiple pro-oncogenic changes in the levels of protein expression and function. Genes most commonly associated with the process of oncogenesis include: p53 inactivating mutation; hDM2 overexpression; p16 reduced expression; K-/H-RAS activating mutation; PTEN inactivating mutation/deletion; EGFR activating mutation and overexpression; retinoblastoma inactivating mutation and deletion; Cyclin proteins overexpression; CD95 reduced expression; protective BCL-2 proteins overexpression; to name but just a few of such molecules.^1-5 That the minimally required specific proteins for oncogenesis are not known for many specific tumor types remains a challenge for the rational design of molecular targeted therapies.

Keywords: :

• basal-like breast cancer
• EGFR
• PTEN
• p53
• MCF10A
• mammary cells
• oncogene
• tumor suppressor
• transformation
• soft agar

This article refers to:

Alterations of EGFR, p53 and PTEN that mimic changes found in basal-like breast cancer promote transformation of human mammary epithelial cells

In the manuscript by Pires et al., the role of proteins whose function is known to be altered in basal-like breast cancer cells were investigated in an immortal though not tumorigenic mammary tumor cell line, MCF10A.⁶ MCF10A is particularly useful for the present studies as based on expression profiling studies it expresses markers most often associated with the basal-epithelial phenotype.⁷ The authors chose to examine three well described proteins whose functions are altered in basal breast cancer cells; PTEN deletion; p53 mutant inactive; EGFR (also called ERBB1) mutant active. As individual oncogenes, only deletion of PTEN could stimulate 2-dimensional MCF10A cell growth in the absence of exogenous growth factors; and, the effect of a single PTEN deletion was not replicated when cells were grown in a 3-dimensional culture in soft agar. Interestingly, expression of H-RAS V12 in MCF10A cells did facilitate 3D culture growth; as the H-RAS V12 point mutants specific for activation of ERK/AKT/RAL were not used in this study it is not known which specific downstream pathways are required for this 3D growth effect.^8,9 The authors then performed analyses using double and triple mutant MCF10A cells. In 2D culture all double mutants except the EGFR + p53 mutant grew robustly. Similar data were obtained when these cells were grown in 3D culture. Of note was that the triple mutant (p53 + PTEN + EGFR) grew more rapidly and formed more colonies than either of the matched double mutants. This supports the multi-hit hypothesis in the MCF10A line. Nevertheless, the triple modified cells were unable to form tumors in SCID mice after 3 mo, suggesting that still more mutations are required for in vivo growth. Whether the H-RAS V12 MCF10A cells used in these studies formed tumors was not reported.

A notable difference in signaling between (PTEN + p53) cells and cells expressing the EGFR was further activation of AKT, though this activation of AKT could not explain why (p53 + PTEN + EGFR) grew more rapidly and formed more colonies. A more significant difference was that (p53 + PTEN + EGFR) cells expressed higher protein levels of EGFR and had higher phospho-EGFR levels. Whether (p53 + PTEN + EGFR) cells also express more paracrine EGFR ligands, e.g., TGFα; HB-EGF, was not reported. Collectively the data in the Pires et al. study argue that multiple pathways downstream and upstream of RAS are required to fully transform the basal-cell like cell line MCF10A.

Acknowledgments

PD is funded by R01 DK52825.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.